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RAS and CAP Genes (Signal Transduction/Onogenes/Vesicular Tafficking/Camp) JEFFREY E

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RAS and CAP Genes (Signal Transduction/Onogenes/Vesicular Tafficking/Camp) JEFFREY E Proc. Natl. Acad. Sci. USA Vol. 89, pp. 4338-4342, May 1992 Cell Biology SNCI, a yeast homolog of the synaptic vesicle-associated membrane protein/synaptobrevin gene family: Genetic interactions with the RAS and CAP genes (signal transduction/onogenes/vesicular tafficking/cAMP) JEFFREY E. GERST*, LINDA RODGERSt, MICHAEL RIGGSt, AND MICHAEL WIGLERtI *Department of Cell Biology and Anatomy, Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029-6574; and tCold Spring Harbor Laboratory, P.O. Box 100, Cold Spring Harbor, NY 11724 Contributed by Michael Wigler, February 7, 1992 ABSTRACT SNCI, a gene from the yeast Saccharomyces MATERIALS AND METHODS cerevisiae, encodes a homolog of vertebrate synaptic vesicle- associated membrane proteins (VAMPs) or synaptobrevins. Microbial Culture. Yeast strains were grown in rich medium SNCI was isolated by its ability to suppress the loss of CAP (YPD; yeast extract/Bactopeptone/dextrose), synthetic com- (SC), or SC drop-out minimal medium function in S. cerevisiae strains possessing an activated allele of plete minimal medium lacking an essential amino acid or nucleotide base. Drop-out is a ofthe RAS-responsive S. cerevisiae RAS2. CAP component minimal medium was used to maintain selection of plasmids. adenylyl cyclase complex. The N-terminal domain of CAP is Yeast extract, Bactopeptone, and yeast nitrogen base lacking required for full cellular responsiveness to activated RAS ammonium sulfate and amino acids (YNB) were purchased proteins. The C-terminal domain of CAP is required for from Difco. YPD was prepared according to Sherman et al. normal cellular morphology and responsiveness to nutrient (17). SC minimal complete and drop-out media were prepared extremes. Multicopy plasmids expressing SNCI suppress only as described by Sherman et al. (17) and consisted of0.7% YNB the loss of the C-terminal functions of CAP and only in the supplemented with the appropriate auxotrophic requirements presence of activated RAS2. and 2% glucose. Yeast medium lacking in amino acids and a nitrogen source (YNB-N) was prepared according to Toda et The yeast Saccharomyces cerevisiae contains two RAS al. (4). Standard methods were used to introduce plasmids into genes that encode proteins highly homologous to mammalian the various yeast strains (17). Escherichia coli strains HB101 RAS oncogene products (1, 2). These RAS proteins are and DH5a were used for plasmid transformations and plasmid required to activate S. cerevisiae adenylyl cyclase (3, 4) but DNA preparations. may have other functions as well (5). The functions of RAS Yeast Strains. cap yeast strains SKN32 (Mata leu2 ura3 in higher organisms are not known. When expressed in S. trp) ade8 can) cap::HIS3) and SKN37 (Mata leu2 ura3 trp) cerevisiae, mammalian RAS proteins are capable of both ade8 can) RAS2V1l19 cap::HIS3) have been described (9). activating adenylyl cyclase and suppressing the lethality This cap:.HIS3 allele lacks amino acids 78-451 ofthe coding associated with the loss ofendogenous RAS function (3, 5, 6). region of CAP and is a null allele. cap strain SK013 (Mata Thus, some functions of RAS may have been conserved leu2 ura3 trp) ade8 can) cap::HIS3) has also been described during the course ofevolution. To explore this we have begun (9). cap strain SKN50 (Mata leu2 trp) ade8 can) iral::HIS3 to characterize the S. cerevisiae adenylyl cyclase complex. cap:: URA3) was created by transforming the iral strain IR-1 We previously identified a protein called CAP that copu- (18) with the EcoRI fragment of the CAP disruption plasmid rifles with a RAS-responsive adenylyl cyclase complex (7). pUSMN2 as described (9). cap strains SKN55 and SKN56 The gene for CAP encodes a 526-residue protein that is (Mata ade8 can) bcy)::LEU2 tpk2::HIS3 tpk3::TRP) required for full cellular responsiveness to activated RAS and cap:: URA3) were created by transforming the bcy) tpk2 tpk3 for normal cellular morphology and responsiveness to nutri- strain S13-58A (19) with the EcoRI fragment of pUSMN2 ent extremes (8, 9). Deletion analysis has shown that CAP is CAP disruption plasmid. cap strains SKN58 and SKN59 bifunctional (10). Expression of a domain consisting of the (Mata ade8 trp) can) pdel::LEU2 pde2::URA3 cap::HIS3) N-terminal 168 amino acids is sufficient for full cellular were created by transforming the pdel pde2 strain DJ23-3C responsiveness to activated RAS, while expression of the (20) with the EcoRI fragment of the CAP disruption plasmid C-terminal 160 amino acids is sufficient for normal cellular pHSPN5 as described (9). HIS3+ transformants were isolated responses to nutrient extremes (10). At present, it is unclear and the disruption of CAP was verified by phenotypic and whether RAS or adenylyl cyclase influences CAP function. Southern blot analysis. The snc) strain JG4 (Mata leu2 trp) To understand the function ofCAP, we have isolated genes ade8 his3 can) snc):: URA3) was constructed by transform- that on multicopy plasmids are capable of suppressing loss of ing the haploid SP1 yeast strain (Mata leu2 ura3 trp) ade8 C-terminal function. One such gene, PFY, encodes profilin, his3 can)) (4) with the Sal I/Sac I fragment of the SNCI an actin binding protein (11). Another gene, which we have disruption plasmid pORF3U (see below). Integration of this named SNC) (suppressor of the null allele of CAP), is construct at the SNC) locus results in an insertion in SNC). described here. It encodes a protein homologous to low The snc) strain JG5 (Mata leu2 trp) ade8 his3 can) snc)A::URA3) was created by transforming the haploid SP1 molecular weight proteins known as VAMPs (synaptic ves- yeast strain with the Sal I/Sac I fragment of the SNC) icle-associated membrane proteins) (12, 13) or synaptobre- of this vins (14-16) that are associated with synaptic vesicles and are disruption plasmid pNCSU (see below). Integration found in a wide variety of organisms.§ Abbreviations: VAMP, synaptic vesicle-associated membrane pro- tein; ORF, open reading frame. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" §The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M91157). 4338 Downloaded by guest on September 29, 2021 Cell Biology: Gerst et al. Proc. Natl. Acad. Sci. USA 89 (1992) 4339 construct at the SNCJ locus results in deletion of the SNCI pADH-CAPA15, which expresses 283 amino acids of the N gene. Diploid yeast, formed by mating DC124 (Mata leu2 terminus of CAP (10); and YEpIRA2, which expresses IRA2 ura3 trpl ade8 his4) (19) and SP1, were also transformed with (R.-M. Ballester and M.W., unpublished results). the Sal I/Sac I fragment of pORF3U. Ura' transformants Other plasmids included the following: YEpSNC1, a were sporulated and subjected to tetrad analysis (17). Dis- YEp13M4 plasmid bearing a 3.4-kilobase (kb) Sau3A partial tribution ofthe URA3 marker in haploid strains derived from digestion fragment of genomic SNC1; YEpTSNC1, a pTV3 tetrad analysis was found to be 2:2. In all the cases described plasmid bearing this gene as a Sal I/Sac I fragment; pUC- above, genotypes were verified by Southern blot analysis. SNC, a pUC118 plasmid bearing this fragment; pADH-SNC1 DNA Manipulations. DNA restriction endonucleases, Taq and pADH-cSNC1, which contain a 550-base-pair (bp) frag- polymerase, and T4 DNA ligase were used as recommended ment ofgenomic SNC1 or a 370-bp fragment ofSNCI cDNA, by the suppliers (New England BioLabs and Cetus). Molec- cloned into the Sal I and Sac I sites of pAD4A, respectively; ular cloning, Southern blotting, and colony hybridization pADH-ASNC1, which bears a 2.3-kb BamHI fragment, con- techniques were performed as described by Maniatis et al. taining the ADHI promoter and the SNCJ sequence from (21). DNA sequencing was performed by the dideoxynucle- pADH-SNC1, cloned into the BamHI site of pAV3; and otide chain-termination method (22). The polymerase chain pADH-ACAPA4, which bears a 3.0-kb BamHI fragment, reaction (PCR) (23) and subcloning of PCR products were containing the ADHI promoter and the CAPA4 allele (10) carried out as described (10). Oligonucleotides used to am- from pADH-CAPA4, cloned into the BamHI site of pAV3. plify SNCJ included a forward oligonucleotide bearing a Sal The modified pAV3 plasmids were used in the transformation I site (5'-AACGTATTCGTCGACCATGTCGTC-3') and a of cap strains SKN55-56 and SKN58-59. reverse oligonucleotide bearing a Sac I site (5'-CTA- Two plasmids derived from pUCSNC were used for dis- CATATGGGAGCTCCCTAT-3'). Total RNA was isolated ruptions of SNCI: pORF3U, which has the URA3 selectable from wild-type yeast (SP1) according to Sherman et al. (17). marker cloned into the Sty I site (base pair 325) of SNC1; and Isolation ofpoly(A)+ RNA was accomplished with a kit from pNCSU, which has URA3 cloned into the Spe I sites, which Stratagene. First-strand cDNA synthesis from yeast flank SNCJ (base pairs -237 and +646, respectively). For poly(A)+ RNA, which was used as a template for PCR, was pNCSU, pUCSNC was digested with Spe I, which removes accomplished with a cDNA synthesis kit from Bethesda the entire SNCJ coding region, and the URA3 gene was Research Laboratories. cloned into this site. Both disruption constructs were verified Genomic DNA from a YPD' revertant of the SK013 cap by restriction analysis. mutant strain (9) was isolated according to Sherman et al. (17). The DNA was partially digested with the Sau3A re- RESULTS striction endonuclease and size-fractionated by gel electro- A Yeast Gene That Suppresses Loss of the C-Terminal phoresis.
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